1st field of activity
Prof. Dr. Kay Hamacher (TU Darmstadt, Biologie)
Starting from established methods and results on Ku70/80 as basis for radiation damage response (previous students S. Knorr and S. Weissgraeber) we will leverage recent results on the molecular-structural basis of the PkCs-Ku-Interaction (PhD student M. Schmidt). These will stear coarse-grained simulations of the full complex of DNA/Pkcs/Ku-complex to understand the underlying mechanism. Furthermore we are interested in the functional-structural basis of additional repair proteins (such as Nek1, Rad54, …).
Dr. Alexander Rapp (TU Darmstadt, Biology)
BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing)  is a novel method to identify DSBs with a base pair resolution in the genome wide context. This method uses in situ ligation of sequencing adaptors to subsequently purify sequence tags and map DSBs to their genomic location. So far this method has not been used in radiation biology. In this project the BLESS method will be used together with ɣH2AX ChIP-seq approaches we successfully established in the 1st and 2nd generation of the GrK to study the distribution of DSBs together with the corresponding ɣH2AX signal. This allows a direct correlation between the DSB location and the corresponding ɣH2AX spreading. Since the BLESS methods involves a complex DNA end repair step, the method can be modified to allow the discrimination between simple (directly repairable DSBs) as well as complex DSBs that require different steps of end repair in situ. Furthermore the BLESS-adaptor will be used to visualize DSBs directly in situ by super-resolution microscopy together with HR and NHEJ factors to discriminate between different repair pathways and correlate this to the location of the DSB in the cell nucleus as well as to the genomic environment.
Prof. Dr. M. Cristina Cardoso (TU Darmstadt, Biologie)
To ensure the maintenance of the genetic and epigenetic information the cell must precisely duplicate its chromosomes every time it divides. This duplication starts at multiple chromosomal sites (origins) proceeding bidirectionally by the coordinated action of a large catalog of enzymes and auxiliary factors constituting the replisome. How these activities are coordinated in vivo and how the whole chromosomes get completely and correctly duplicated albeit encountering thousands of different lesions along the way is unclear.
Our aim is to understand the mechanisms coordinating and structurally organizing in space and time the multiple steps of this process at the level of the individual molecules, single replicons as well as the entire genome and relate that with the repair of DNA damage. We want to measure the interactions and live-cell kinetics of different components of the replication/repair machinery in mammalian cells using advanced fluorescence microscopy techniques. Importantly, we want to visualize and quantify replicons and repair domains and their interrelationship with chromosomal organization using super-resolution fluorescence microscopy techniques. Furthermore, we want to investigate how the replisomes and replicons respond to DNA lesions induced by radiation stress and how that is affected by their 3D distribution within the cell nucleus and local chromatin (im)mobility.
Dr. Burkhard Jakob (GSI, Biophysik)
The therapeutic window in radiation therapy is rather defined by limiting normal tissue toxicity and not by dose delivered to the tumour region. Radioprotectors are compounds that are designed to reduce the damage in normal tissues caused by radiation. These compounds are often antioxidants and must be present before or at the time of radiation for effectiveness. Other agents, termed mitigators, may be used to minimize toxicity even after radiation has been delivered. They act by the activation of signalling cascades, alteration of gene expression and/or influencing the energy and redox balance of the cell. However for several radiation mitigators under preclinical consideration, the molecular mechanism of their activity is not well understood. Especially, there is a lack of knowledge if also carbon ion therapy can benefit from combination with radiation mitigators, as high LET irradiation yields more complex damage due to the inhomogeneous dose distribution.
In the proposed project, the effect of radiation mitigators targeting the metabolism or radiosensitsier promoting chromatin compaction should be analysed on their effect on DNA DSB repair. The main emphasis will be put on live cell experiments which include real time recruitment and repair studies comparing x-ray and charged particles under oxic or hypoxic conditions to mimic different tumour environment. These measurements will be complemented by sophisticated microscopic techniques like Fluorescence Lifetime Imaging Microscopy (FLIM) or Fluorescence Recovery After Photobleaching (FRAP) to determine the metabolic status, the chromatin compaction as well as protein-protein interaction and protein binding in living cells after treatment with the drugs and/or radiation.
The study should lead to a better understanding of molecular details of the mitigation effect as well as the influence of radiation quality on the achievable protective or, in case of radiosensitisers, damage promoting effects.
Prof. Dr. Markus Löbrich (TU Darmstadt, Biologie)
Homologous Recombination (HR) is a well characterized DNA double-strand break (DSB) repair pathway which requires the presence of a sister chromatid to restore the lost genomic sequences at the break site. This prerequisite restricts HR to the late S and G2-phases of the cell cycle and makes it a highly error free repair pathway. Thus, HR plays an important role in the maintenance of genomic stability. Many studies performed in yeast have unveiled the importance of Rad52 in HR. Absence of Rad52 renders yeast cells highly radiosensitive, results in impaired DNA repair and proves to be lethal. On the other hand, depletion of Rad52 in mammalian cells shows very mild phenotypes and does not have any significant effect on the repair of DSBs. One reason for this observation is that BRCA2, a key factor of HR, might have overtaken the function of Rad52 in higher eukaryotes.
A recent study sparked new interest to study Rad52 function as it showed that knocking out Rad52 in BRCA2 deficient tumor cells, derived from hereditary breast cancer patients, leads to synthetic lethality. This property can be explored to specifically kill tumor cells without having any adverse effects on healthy cells/tissues. Unfortunately, the mechanisms underlying the phenomena of synthetic lethality are very poorly understood. Work from our lab (AG Löbrich) has shown that depletion of Rad52 in BRCA2 deficient tumor as well as fibroblast cell lines results in the activation of an alternative DSB repair pathway which gives rise to increased chromosomal fusions and that, we speculate, underlie the induced synthetic lethality.
Our aim is to better characterize the physiological function of Rad52 in the repair of ionizing radiation induced DSBs in healthy as well as tumor cells. We are interested to understand the importance of Rad52 in BRCA2 deficient tumor cells and the mechanisms underlying the cause of synthetic lethality observed in Rad52 + BRCA2 double knock out cells. To this end, we would like to deploy various laboratory techniques such as live cell microscopy, immunofluorescence analysis, co-immunoprecipitation studies and also generate Rad52 mutant and knock out cell lines. In the future, we are also planning to collaborate with the group of Prof. Dr. Franz Rödel at the University Hospital Frankfurt in order to perform animal experiments to test different Rad52 inhibitors which can be potentially used for cancer treatment.
PD Dr. Tobias Meckel (TU Darmstadt, Biologie)
It is now well accepted that the microenvironment of cells has a profound impact on their physiology which traditional two-dimensional cell culture methods are unable to provide. To this end, we have developed a collagen based cell culture system that allows us to apply the single molecule microscopy toolkit to cells in a 3D environment (Lauer at al. 2014). Using our system, we aimed to identify the molecular origin behind “cell adhesion mediated radio resistance (CAM-RR)” by following the nanoscale distribution of key adhesion receptors (integrin beta1 and beta3) in irradiated cells cultured under both conditions. We found a strong correlation between the nanoscale distribution of integrin beta1 and the level of phosphorylated FAK in dependence of both culture conditions and doses of X-irradiation. Integrin beta3, in turn, did not show this clear correlation. Cells cultured in a 3D environment were able to maintain a well-organized cluster status of integrin beta1 up to ionizing radiation doses of 15 Gy, whereas cluster disintegration in 2D cultured cells became evident already at 2 Gy (Babel et al. 2016/2017).
Clearly, the strong correlation of our results with the culture conditions suggest an involvement of mechanosensitive mechanisms, i.e. the transmission of mechanical signals through direct molecular connections between integrins and chromatin via cytoskeletal filaments and nuclear scaffolds (Fedorchak et al. 2014). In addition, our observations show that cells have an impaired capacity to maintain clustered integrins after exposure to ionizing radiation.
Hence, the imminent question is, how mechanosensing between integrins and chromatin controls the integrin cluster status and how ionizing radiation is able to interfere in this process. While it is well known that outside-in forces, i.e. the sensing of mechanical properties of the ECM, triggers the assembly of focal adhesions, much less is known about the contribution of cell-generated forces. Based on our results, we hypothesize that chromatin remodeling in response to ionizing radiation impairs the force propagation from the nucleus to integrins which, under normal conditions, is required to maintain their cluster status. A prominent target which may be involved in this process is the ataxia telangiectasia and Rad3-related (ATR) protein, which (i) is recruited and activated by DNA double-strand breaks, (ii) phosphorylates several key proteins that initiate the activation of the DNA damage checkpoint leading to cell cycle arrest, DNA repair, or apoptosis, and (iii) has been identified to be involved in chromatin organization in response to mechanical cues (Kumar et al. 2014). Hence, ATR-deficient cells will be cultured in 3D environments in order to test their ability to maintain integrin beta1 clusters after X-irradiation. In addition the entire force-propagation-pathway between integrins and chromatin (talin, vinculin, actin, nesprin, SUN 1/2, lamin A/C and lamin B) will be addressed in our study to link DNA damage with the nanoscale distribution of integrins.